Project Summary. E. coli RNA polymerase is the multi-subunit molecular machine that catalyzes transcription. It first acts as a physical machine to bind and bend promoter DNA, placing the double stranded DNA into the active site cleft and opening it to form the transcription bubble. Subsequent conformational changes are made to the open complex to provide extra stability and facilitate transcription. RNA polymerase then acts as a chemical machine by catalyzing phosphodiester bond formation, synthesizing an RNA chain. The mechanism of transcription by RNA polymerase is highly conserved across organisms and is tightly regulated in cells; open complexes capable of initiating transcription have promoter-dependent lifetimes that drastically differ by a factor of 104 (?PR: 13 hours; rrnB P1: 1 second) These differences are found to be sequence dependent in the discriminator region of DNA, a 6-8 base pair sequence that lies directly upstream of the transcription start site. However, the purpose of these varying stabilities at the open complex and the role these lifetimes play in transcription are currently not known. This research focuses on the characterization of polymerase/DNA interactions within the open complex to determine their crucial role in the initiation of RNA synthesis and promoter escape. To characterize the promoter/polymerase interactions in transcription initiation, research is aimed to 1) quantify fast kinetics of initiation and the time-dependent distribution of short and long RNAs; 2) Examine whether initiation kinetics, short/long RNA distribution correlate with open complex lifetime; and 3) Compare initiation kinetics and RNA product distribution for RNAP variants affecting open complex lifetime with WT RNAP. To accomplish these aims, novel discriminator-swapped promoter DNA is used in parallel with wild type promoter sequences, and wild type RNA polymerase is compared to site-specific mutant RNA polymerase in a series of dissociation assays, chemical footprinting experiments, and single-round transcription assays. Further, these experiments rely on filter binding techniques, acrylamide gel electrophoresis and densitometry quantitation, and Matrix-Assisted-Laser-Desorption-Ionization Mass Spectrometry to obtain dissociation rate constants, open complex structural conformations and lifetimes, absolute lengths and sequences of RNA transcript products, and transcription initiation kinetic data on a millisecond to second timescale. Data collected will offer a clear picture of the promoter/polymerase determinants in the relationship between open complex lifetime and transcription initiation. These findings will provide new insight in cell proliferation at the level of transcription, expand our fundamental knowledge on gene regulation, and afford new opportunities for antibiotic development.